Agron. Mesoam. 29(2):263-274. Mayo-agosto, 2018 ISSN 2215-3608, doi:10.15517/ma.v29i2.30424 http://www.revistas.ucr.ac.cr/index.php/agromeso
An alternative inoculation technique of Colletotrichum gloeosporioides on mango for early anthracnose tolerance screening1 Una técnica alternativa de inoculación de Colletotrichum gloeosporioides en mango para la detección temprana de la tolerancia a la antracnosis Abraham Monteon-Ojeda2, José Sergio Sandoval-Islas2, José Antonio Mora-Aguilera2, Carlos De León-García De Alba2, Amado Pérez-Rodríguez2, Alfonso Vásquez-López3
Abstract The importance of having a technique that allows an efficient expression of symptoms of anthracnose is based on the early differentiation of cultivars and the optimization of genetic, material and financial resources. The objective of this research was to generate an alternative inoculation technique for Colletotrichum gloeosporioides on mango for early anthracnose tolerance screening. On this technique was optimized some of the most relevant components such as isolate virulence, conidial density, the inoculum deposition on leaves and using of surfactants. The study was carried in Iguala, Mexico, during the production cycles 2015-2016. C. gloeosporioides was biologically and culturally characterized. Gro and Sin monosporic strains were isolated from leaves, flowers, fruits and branches with anthracnose symptoms from commercial mango orchards located in Guerrero and Sinaloa states, Mexico. These strains show mycelial growth at 2.2 and 2.1 cm of diameter per day, spore density of 4.3x106, 3.9x106 conidia/ml, germination of 27 and 26%, virulence, with incubation period 4.5 and 4.1 days after inoculation, incidence of 90 and 92% and severity of 3.2 and 3.5 cm of diameter. The highest incidence and severity values with the lowest incubation period, was obtained using Gro isolate (1x105 conidia/ml) and polyoxyethylene-20-sorbitan monolaurate as spreader-sticker inoculated on the abaxial surface on detached young leaves, 15-20 days old, with a soft brush and incubated under dark condition. This inoculation technique allowed the optimal expression of C. gloeosporioides virulence in mango leaves and could be incorporated as a tool in the early differentiation of tolerance and susceptibility among cultivars. Keywords: conidial, spores, Mangifera indica, isolation techniques.
Resumen La importancia de contar con una técnica que permita una expresión eficiente de síntomas de antracnosis se basa en la diferenciación temprana de cultivares y a la optimización de recursos genéticos, materiales y financieros. El objetivo de esta investigación fue generar una técnica de inoculación alternativa para Colletotrichum gloeosporioides Recibido: 6 de setiembre, 2017. Aceptado: 15 de enero, 2018. This work was part of first author´s doctoral thesis, carried on the Colegio de Postgraduados, Mexico. 2 Colegio de Postgraduados, Instituto de Fitosanidad. Km 35.5, Carr. Mexico-Texcoco, Montecillo, Mexico State, C. P. 56230, México.
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[email protected], perez.amado@ colpos.mx 3 Instituto Politécnico Nacional, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (CIIDIR Unidad Oaxaca); Hornos 1003, Col. Noche Buena, Santa Cruz Xoxocotlán, CP 71230, Oaxaca, México.
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Monteon-Ojeda et al.: Alternative inoculation technique of mango anthracnose
en mango para la detección temprana de la tolerancia a la antracnosis. En esta técnica se optimizaron algunos de los componentes más relevantes como la virulencia de los aislamientos, la densidad de conidios, la deposición de inóculo en hojas y el uso de surfactantes. El estudio se realizó en Iguala, México, durante los ciclos de producción 2015-2016. C. gloeosporioides fue caracterizado biológica y culturalmente. Los aislamientos monospóricos Gro y Sin, fueron obtenidos de hojas, flores, frutos y ramas con síntomas típicos de antracnosis procedentes de huertos comerciales de mango ubicados en los estados de Guerrero y Sinaloa, México. Estos aislamientos presentaron un crecimiento micelial de 2,2, y 2,1 cm de diámetro por día, densidad de esporas de 4,3x106, 3,9x106 conidios/ml, germinación de 27 y 26%, virulencia, con un período de incubación de 4,5 y 4,1 días después de la inoculación, 90 y 92% de incidencia y severidad de 3,2 y 3,5 cm de diámetro. Los valores de incidencia y severidad más altos y los períodos de incubación más bajos se obtuvieron con el aislamiento Gro (1x105 conidios/ml), en un medio de monolaurato de polioxietileno20-sorbitán sobre la superficie abaxial de hojas jóvenes desprendidas, de 15-20 días de desarrollo, con el empleo de una brocha de pelo suave e incubado en condiciones de oscuridad. Esta técnica de inoculación permitió la expresión óptima de virulencia de C. gloeosporioides en hojas de mango y podría incorporarse como una herramienta en la diferenciación temprana de la tolerancia y susceptibilidad entre cultivares. Palabras clave: conidios, esporas, Mangifera indica, técnicas de aislamiento.
Introduction Mexico is the main mango exporter worldwide and placed among the first five producer countries (FAO, 2016). Anthracnose is one of the most important diseases of this crop, mainly caused by Colletotrichum gloeosporioides (Penz.) (Ploetz and Prakash, 2000). C. gloeosporioides has cosmopolitan distribution in mango producing regions, affects most severely in flowering, fruit setting, and postharvest (Carreon et al., 2010). The initial symptoms in leaves consist of small dark spots with chlorotic halo; lesions can grow and coalesce to reach ± 1.0 cm in diameter. Flowers show small brown lesions on the primary and secondary axis that result in a blight of the panicle (Prusky, 1994). Anthracnose cause losses ranging from 50 to 100% and severity from 70 to 80% in young fruits (8-15 mm), with high environmental humidity and inadequate agronomical management (Arauz, 2000; Prusky et al., 2009). An inoculation technique of C. gloeosporioides that permit to determine optimal conditions for infection, development of symptoms and to study the relationship of the pathogen with their hosts may be useful for testing resistance in new varieties of mango and to establish strategies to manage this disease (Denoyes-Rothan et al., 2003; Hernández et al., 2005; Moral and Trapero, 2009). Most of the studies on etiology and epidemiology of C. gloeosporioides, report inoculation methods of the pathogen in flowers and fruits; however, research on inoculation techniques of leaves is limited (Acosta et al., 2001; Biggs and Miller, 2001; Gutiérrez-Alonso et al., 2003; Moral and Trapero, 2009) and operates with different efficiency degrees, often being inconsistent. These protocols generally consider several components of a technique frequently applied in separate form as inoculum densities and deposition methods, additives, phenological stages and incubated conditions (Paéz, 1997). The objective of this research was to generate an alternative inoculation technique for C. gloeosporioides, on mango for early antracnose tolerance screening.
Materials and methods Study site The study was carried out in the Unidad Académica de Ciencias Agropecuarias y Ambientales of the Universidad Autónoma de Guerrero, in Iguala, Guerrero, Mexico (18° 25’N, 99° 35’W, 731 masl); during the 264
Agron. Mesoam. 29(2):263-274, mayo-agosto, 2018 ISSN 2215-3608 doi:10.15517/ma.v29i2.30424
Monteon-Ojeda et al.: Alternative inoculation technique of mango anthracnose
production cicles 2015-2016. The mango plants were 18-month-old, grafted with “Ataulfo” cultivar, established in plastic pots in a nursery covered with transparent plastic (caliber 600) and a polypropylene mesh, 50% shade. The plants were fertilized by applying in a weekly basis Steiner nutrient solution (1.0 l per plant poured in the soil and 1.0 ml/l sprayed to the canopy) and watered to field capacity every third day. Temperature, relative humidity and photoperiod in the nursery were registered every two hours with a Hobo® data logger, model U12. Strains obtainment and culture characterization Leaves, flowers, juvenile fruits and annual vegetative branches with anthracnose symptoms were collected from commercial mango orchards located in the states of Oaxaca, Guerrero, Colima and Sinaloa, Mexico. Plant tissue fragments, approximately 1 cm long with typical symptoms, were disinfected with sodium hypochlorite 1% for 2 min, rinsed three times with sterile water and dried with a sterile absorbing towel before being transferred to Petri plates containing potato-dextrose-agar medium. Plates were incubated at 28 °C for seven days under alternating 12:12 h light-dark conditions. To evaluate growth rate, sporulation and germination, a 2 cm diameter PDA disc from each strain was transferred to individual plates with PDA and incubated at 22-25 ºC during seven days. The diameter of the colony was measured (cm) with a digital vernier. Five replicates were done per isolate (treatment). To estimate sporulation, five 2 cm diameters mycelial, were collected per isolate from seven-day-old cultures and placed in a blender with 50 ml sterile distilled water, and mixed during four 2 s intervals to promote conidia and acervuli detachment. The content filtered through a metallic mesh (200 mesh/in2) was collected into a glass beaker and adjusted to 100 ml with sterile distilled water. The conidial suspension was vortexed for 20 s and concentration of conidia/ml was estimated in five preparations using a Neubauer Chamber. The spore viability was quantified by depositing one drop (50 µl) of inoculum (1x105 conidia/ml) in an excavated slide with cover slide and placed in a Petri plate on a wet sterile paper disc to avoid dehydration. Slides were incubated at 24 ± 3 °C under 12:12 light and dark conditions. Was considered as some germinated conidia when these emitted a germinative tube longer than his body, and were quantified after 48 h through light microscopy. Five slides (experimental units) were quantified per isolate (treatment) in each condition. Additives and conidial viability Gro isolate at a density of 1x104 conidia/ml was mixed with 0.1% of polyoxyethylene-20-sorbitan monolaurate, 0.1% isoparaffin commercial mixture of ethoxylated polyglycol and 0.1% aryl-polyethoxy ethanol alcohols. Sterile distilled water was included as control. One drop of each suspension was placed in an excavated slide, covered and placed in a Petri plate on a wet sterile paper disc to avoid dehydration, and incubated at 24±3 ºC in continuous darkness. Conidial germination was quantified at 24 and 48 h through light microscopy. Isolates virulence in detached leaves The incubation period (Pi), incidence and severity of each isolate were evaluated using the next detached leaf inoculation: vegetative buds were marked in 18-month-old cv. “Ataulfo” plants in the nursery to observe foliar development; the leaves were cut when they were 20-days-old, then were disinfested with sodium hypochlorite at 0.5% for 30 s, rinsed three times with sterile distilled water and blotted in a laminar flow cabinet in asepsis conditions. Previously, 25 x 35 x 10 cm (L, W, D) plastic containers were disinfested with sodium hypochlorite (5%) and cleaned with alcohol (95%), the containers were left in a laminar flow cabinet until dry. Then, the bottom of the container was covered with sterile towels humidified to saturation with sterile distilled water. Agron. Mesoam. 29(2):263-274, mayo-agosto, 2018 ISSN 2215-3608 doi:10.15517/ma.v29i2.30424
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The disinfested and dried mango leaves were placed on the wet towels inside the plastic container. Half of each leaf (respect to the central nerve) was inoculated by depositing (without wounds) three drops (50 ml) of a conidial suspension (1x105 conidia/ml) on the adaxial and abaxial surface (separately) of the leaves. Each drop was placed separately, equidistant, approximately 35 mm from the central nerve of each leaf to generate individual lesions. The plastic containers with the inoculated leaves were covered with transparent polyethylene and incubated at 24 ± 3 ºC, under alternate 12:12 dark: light conditions and 100% relative humidity for eight days. Five leaves were placed in each container (experimental units), and five containers per strain (treatments) were evaluated. The Pi and incidence were quantified. The severity was determined eight days after inoculation (dai) measuring each lesion diameter (cm) with a digital vernier. Inoculum densities Cv. “Ataulfo” 20-days-old mango leaves were inoculated with Gro isolate due to its high virulence, using 1x104, 1x105, 4x105 and 1x106 conidia/ml with polyoxyethylene-20-sorbitan monolaurate (0.1%) using the detached leaf technique previously described. In this test, the inoculum was deposited only on the abaxial surface of the leave. The plastic containers with the inoculated leaves were incubated at 24±3 ºC under alternate 12:12 dark: light conditions and 100% relative humidity. Five leaves per container (experimental unit) were inoculated and four containers (replicates) by treatment (doses) was evaluated. The Pi and incidence were quantified. The severity was evaluated after eight days, considering the diameter of each lesion (cm) with a digital vernier. Statistical analysis For each one of the trials a completely randomized design was used and analysis of variance (GLM) and mean tests (LSD, p = 0.05) were performed with SAS v.9.3 (SAS Institute Inc, 2012). Plant nursery inoculation Nursery 18-month-old mango plants cv. “Ataulfo” with similar growth and vigor characteristics were selected for this study. Vegetative buds were marked and when the leaves had twenty days of development they were detached and disinfested with 0.5% NaCl solution for 30 s, rinsed three times with autoclaved distilled water using a hand-held sprayer and left to dry for 10 min. Five inoculation procedures were evaluated: 1) manual spraying, 2) contact with a cotton swab, 3) contact with a soft brush (camel hair), 4) contact with cotton cloth, and 5) direct mycelium contact. Inoculation was done on both abaxial and adaxial surface of the leaves inoculating on one half of the leaf, considering the central foliar nerve as reference. A 1x105 concentration of conidia/ml suspended in polyoxyethylene20-sorbitan monolaurate (0.1%) of Gro isolate was used. Five leaves per plant (experimental unit) were inoculated and four plants (replicates) per treatment (inoculation procedure) evaluated. Inoculation was done before sunset at 18:00 h (± 300-450 lm), inoculated leaves were covered with a dark plastic bag during 12 h and the plants were kept in a nursery covered with shading mesh (70% shade) until symptoms appeared. Pi and incidence were determined. Severity was evaluated 15 dai through digital images, estimating the affected area (%) of each leaf using the GIMP 2.0 software for Windows®. In the nursery, the temperature oscillated between 29 and 31 ºC, the relative humidity 85-90%, and the photoperiod 12±1 h light. These variables were registered every two hours with a Hobo® data logger, model U12. 266
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Monteon-Ojeda et al.: Alternative inoculation technique of mango anthracnose
A completely randomized block statistical design was used, and a variance analysis and mean separation (LSD, p≤ 0.05) were done using the SAS v.9.3 (SAS Institute Inc., 2012). Re-isolating strains Vegetative tissues with symptoms of anthracnose collected from the experimental units of both trials (detached and attached leaf technique) were fragmented into pieces of approximately 1 cm in length, isolating was made using a monosporic culture technique and the species was corroborated using taxonomic keys of Ainsworth et al. (1973), Barnett and Hunter (1998) and Bailey and Jeger (1992).
Results Isolates, virulence and susceptible tissue Monosporic strains Gro, Col, Oax, Sin and Tux were isolated from infected mango tissue; Gro and Sin showed the higher growth rate, sporulation density and germination (Table 1). These factors correlated with virulence (Estrada-Valencia et al., 1997; Montesinos-Matías et al., 2011). It was observed that Gro and Sin isolates induced the highest incidence and severity in detached leaves (Table 2) (p
